Polymethoxyflavones represented by the structural formula shown in the following formula (I) are known in the prior art to be abundantly present in citrus plants, and specifically citrus peels.
(In the formula, R1, R2, R3, R4 and R5 each independently represent hydrogen or methoxy.)
In particular, nobiletin (a compound of formula (I) wherein R1, R2, R3, R4 and R5 are all methoxy groups) is a substance with established physiological activity, including an anti-carcinogenic effect (Non-patent document 1).
In the field of foods as well, polymethoxyflavones are attracting attention for their various useful effects that have been discovered, as taste-improving agents and in taste-improving methods (Patent document 1), or as flavor deterioration inhibitor (Patent document 2).
Citrus plant peel oil contains, in addition to polymethoxyflavones, also numerous different kinds of components with widely varying properties (see Non-patent document 2 and Non-patent document 3, for example). For example, it contains volatile components, including hydrocarbon compounds such as terpenes, carbonyl compounds such as aldehydes and esters, or alcohols, and non-volatile components such as waxes and carotenoid pigments that do not volatilize under ordinary conditions, and it is therefore considered difficult to selectively separate polymethoxyflavones by convenient methods.
As prior art methods for producing polymethoxyflavones from citrus plant peel oil there have been proposed a method of hot extraction of a starting material of Rutaceae plant fruit, peel, peel oil, leaves or the like, using an organic solvent such as methanol, ethanol or chloroform, and a method of extraction using a supercritical fluid solvent (Patent document 1, Patent document 2).
In these methods, however, it has not been possible to obtain highly pure polymethoxyflavones from starting materials that contain hydrocarbon compounds, carbonyl compounds, esters and alcohols, such as limonene, linalool, geraniol, nerol, α-terpineol and citral, and other fragrance components, as well as pigment components such as carotenoids, in addition to polymethoxyflavones. Thus, when polymethoxyflavones obtained by conventional extraction methods are to be used as taste-improving agents or flavor deterioration retarders, their uses have been very restricted due to problems such as altered flavor, off-odor and coloration, caused by contaminants in the extract.
In order to obtain highly pure polymethoxyflavones, therefore, prior art techniques have involved loading an extract containing polymethoxyflavones into a column packed with a support such as silica gel, aluminum oxide, alkylsilylated silica gel, allylsilylated silica gel or the like, and performing separation and refining by column chromatography or high-performance liquid chromatography using a developing solvent such as ethyl acetate-hexane or water-acetonitrile. Liquid-liquid partition chromatography using hexane or pentane and aqueous methanol or ethanol is also sometimes used.
However, the polymethoxyflavones obtained by these methods, while having extremely high purity, are difficult to separate and purify as large amounts of polymethoxyflavones in a single procedure. In addition, long periods of time are required to prepare large amounts of polymethoxyflavones, while highly expensive organic solvents and special equipment are also necessary, making it impossible to avoid increased costs, and therefore such methods are considered unsuitable for industrial production. Moreover, these methods employ organic solvents that can have adverse effects on the human body, such as n-hexane, ethyl acetate, tetrahydrofuran and acetonitrile, and therefore the use of the obtained polymethoxyflavones in food products is inadequate from a safety standpoint.
A simple method for separating polymethoxyflavones has been proposed, as a method in which extraction is performed with an alcoholic aqueous solution from the residue after distillation removal of the volatile components from citrus plant peel oil (Patent document 3). This method allows compositions with high methoxyflavone contents to be obtained by a simple procedure from blackish brown distillation tar residue, but while it can be used directly as an aqueous formulation, precipitates are produced over time and the stability is lacking. In addition, the compositions lack general utility as formulations other than aqueous formulations. That is, when the solvent has been distilled off from the alcohol solution, the methoxyflavone composition solidifies into hard form making it difficult to handle, and this has hampered efforts to prepare oil-soluble formulations and powdered formulations.
Residual pesticides are another problem to be considered when separating polymethoxyflavones at high concentrations from citrus plant peel.
Most citrus plants are administered pesticides or antiseptic agents during cultivation or after harvest for purposes of prevention and measures against insect damage or disease, facilitating insect removal or weed removal, and for prolonged storage of fruit. The administered pesticides and antiseptic agents are removed by rinsing the harvested fruit, but portions remain on the peels, and can potentially be extracted into the peel oil when essential oils are pressed from the peels.
Examples of residual pesticides detected on citrus fruits and bananas imported into Japan include organic chlorine-based pesticides (chlorobenzilate and dicofol), organic phosphorus-based pesticides (pyridaphenthion and methidathion), and pyrethroid-based pesticides (cypermethrin and fenpropathrin).
Most pesticides and antiseptic agents are non-volatile components, and can potentially be concentrated during the refining steps for polymethoxyflavones.
As a method for removing pesticides from plant extracts there is known, for example, (1) a method of dissolution in a liquid mixture of a lower aliphatic alcohol and water at a volume ratio of between 10:90 and 80:20, and passing the obtained solution through a column packed with a porous adsorption resin with a most frequent pore radius of 30-120 angstrom, to absorb the residual pesticides in the solution onto the adsorption resin, and then recovering the plant extract from the solution after treatment (Patent document 4). There is also known (2) a method of contacting a plant extract with a carbon dioxide gas fluid in the supercritical to subcritical state to remove the residual pesticides (Patent document 5).
However, since long periods of time are required for passage through the column in method (1) it is not possible to treat large amounts of extract, while highly expensive organic solvents and special equipment are also necessary, making it impossible to avoid increased costs, and therefore such a method is considered unsuitable for industrial production. Moreover, while it is suitable for removal of organic chlorine-based residual pesticides such as BHC and DDT, it lacks general utility, and the extraction components with properties similar to those of the residual pesticides are lost by adsorption, and the extract yield is reduced.
On the other hand, method (2) allows efficient removal of residual pesticides from plant extracts without loss or degradation of the plant extraction components, but it requires special equipment and is therefore very costly.
Different methods known for removal of pesticides from essential oils include (3) a method of contacting with a strong cation-exchange resin or a strong anion-exchange resin (Patent document 6) and a method of contacting with an aqueous alkali solution (Patent document 7).
However, method (3), while allowing selective removal of basic, acidic and neutral organic compounds, lacks general utility in cases of residue of pesticides with various different physical properties.
Thus, no known method has yet been successfully developed for production of polymethoxyflavone compositions from citrus plant peel oils, that can be used for food products without safety concerns, is convenient and economically advantageous, and has general utility, and therefore a new manufacturing method is desired.